1. Field of the Invention
The invention relates to a procedure for the detection of an element in a sample. A transmission electronic microscope is used to measure a first image of the intensities of the sample for an energy-loss domain before the element edge. A second image of the intensities of the sample for an energy loss is measured in the area of the element edge.
2. The Prior Art
This procedure is also termed the xe2x80x9ctwo-windows difference method.xe2x80x9d The difficulty of this method is that the intensity of the background is a function of the energy loss and, therefore, various background intensities need to be determined within the energy window. The background intensity essentially depends on the thickness of the sample point and decreases as energy loss increases. In order to determine the purely element-specific signal, i.e. the element-specific intensity, initially the intensity is measured in the energy-loss domain specific to the element to be detected.
Therefore, a background value in the energy-loss domain is subtracted from these measured values, where the background in the element-specific energy window is a function of the background outside the element-specific energy window. In the well known method, the assumption is made that the intensity of the background in the domain of the energy window is a linear function of the intensity before the energy window.
For many applications, this assumption is approximately justified. However, the way in which the parameters of the linear function are to be calculated is not satisfactorily described in the state of the art. Furthermore, in many cases a linear function does not correspond to the natural facts.
It is therefore an object of the invention to further develop a generic procedure so that the function described can be determined and reproduced in order to calculate a more precise image of the element-specific intensities.
The invention comprises a procedure for the detection of an element in a sample comprising the steps of measuring a first image of the intensities I1p of the sample for an energy loss before the element edge using a transmission electronic microscope. Then a second image of the intensities I2xe2x80x2p is measured for an energy loss in the area of the element edge. The next step is determining the non-element-specific intensities I1 of the sample for an energy loss before the element edge for various points at a reference sample point that does not contain the element. Then the non-element-specific intensities I2 for an energy loss are determined in the area of the element edge are determined. An approximation function I2 (I1) and an image of the element-specific intensities IE is then calculated from these values. The corresponding intensity I2p with the approximation function for every point in the first image with the intensity I1p is calculated, and the difference between the measured intensity I2xe2x80x2p and the calculated intensity I2p as the element-specific intensity IE for the corresponding point in the second image is determined.
In the present invention, the intensity I1 for an energy loss at various points of a reference sample not containing the element is determined before the element edge. The non-element-specific intensities I2 for an energy loss are determined in the area of the element edge. From these values an approximation function I2 (I1) is calculated and an image of the element-specific intensities IE is calculated. Therefore, for every point in the first image with the intensity I1p, the corresponding intensity I2p is calculated from the approximation function and, for the corresponding point in the second image, the difference between the measured intensity I2xe2x80x2p and the calculated intensity I2p is determined as the element-specific intensity IE.
The procedure of the invention allows the function I2 (I1) to be calculated experimentally. This is useful because it allows the removal of the non-element-specific background from the image of the intensities measured in the area of the element edge for an energy loss. Therefore, systematic error is markedly reduced and the background subtraction can be matched to each sample and to all variable parameters that can be set at the microscope.
The intensity pair I1/I2 can be graphically represented so that it can also be easily determined visually whether the calculated approximation function corresponds with the measured values.
It is advantageous for the reference sample to display a course of different sample thicknesses. Since sample thickness affects the background signal most strongly, a course of different sample thicknesses permits the measurement of various background signals and thus the determination of various intensity pairs I1/I2. This course should display no steps and preferably takes the shape of a ramp.
Preferably, the reference sample displays at least the thickness of the sample. This ensures that, for all background signals occurring in the area of the sample, there is a corresponding background signal to be determined from the reference sample.
Reference samples consisting of pure carbon have shown themselves to be suitable. Such reference samples can be produced without difficulty in all electron-microscope oriented laboratories and are mainly suitable for biological samples if they have not subsequently been treated with heavy metals.
A more detailed determination of the background signal may be required. The reason is that, as well as a purely mass-thickness contrast, a further contrast mechanism plays a role in the emergence of an image. It may, for example, be an inhomogeneous concentration of various elements. A contrast resulting from this is termed a xe2x80x9ccompositional contrastxe2x80x9d (CC). For this reason, it can be advantageous for the reference sample to contain at least two elements. Because of the high carbon content in biological samples, carbon is recommended as one of the elements, while the other element should be a heavier element than carbon, e.g. nitrogen, oxygen or sulphur.
However, the preparation of suitable reference samples is difficult. In a reference sample intended to contain oxygen in addition to carbon, for example, almost all the oxygen is lost by evaporation. Furthermore, a heavier element is more suitable for a reference sample. Sulphur can be prepared in different thicknesses only with great difficulty, if at all.
For this reason, dithiouracil (DTU) was used. In DTU, both of the oxygen atoms of the uracil, which is one of the four bases of DNA, are replaced by sulphur atoms. Compounds containing oxygen may, however, also be considered.
In order to reduce noise in the graphs derived from the images, it is suggested that the intensity of each point be measured as the mean value of its environment. Each of the images, for example, can consist of 0141xc3x971024 image points, the intensity value of each image point being replaced by the mean value of the environment of 10xc3x9710 image points. After the calculated function is obtained, however, the original images are used in further work.
In order to determine the quality of the approximation function and, where appropriate, replace the approximation function with a further approximated function, it is suggested that the quality of the function I2 (I1) be determined by statistical functions. This also permits the reproducible determination of error in the procedure.
The procedure of the invention was tried with great success on the element phosphorus. The procedure can also, however, be used for the detection of other elements, e.g. iron. Since DNA contains phosphorus, the procedure of the invention permits the course of DNA in, for example, viruses or other DNA protein complexes to be shown.
If a residual contrast remains after the procedure of the invention has been carried out, at least a third image of the intensities I3xe2x80x2p of the sample for a third energy loss can be recorded. This energy loss should also lie before the element edge and should differ from the energy loss of the first image. Third intensities I3p for the third energy loss for the various points are then determined at the reference sample point. From the determined first and third intensities I1, I3, the approximation function I2 (I1,I3) is then calculated. The image of the element-specific intensities IE is calculated, in that, for each point of the first and third image, the corresponding intensity I2 is calculated from the approximation function I2. For the corresponding point in the second image, the difference between the measured intensity I2xe2x80x2p and the calculated intensity I2p is determined as the element-specific intensity IE.
The carrying out of such a procedure makes it possible to detect two contrast mechanisms. To make such detection possible without difficulty, the reference sample should be made in such a way that, besides the previously described change in thickness, it also displays an overlap between the two components of the reference sample, e.g. pure carbon and DTU. This can be achieved if carbon and DTU are evaporated in sequence, a mask being displaced between the evaporation processes. The ramps of the two elements are similarly displaced, so that all mass thicknesses and concentration differences in the reference sample can be found.
Expressed in general terms, the calculation of the image of the element-specific intensity IE is given by the following formula:       I    E    =                    I                              2            xe2x80x2                    ⁢          p                    -              I                  2          ⁢          p                      =                  I                              2            xe2x80x2                    ⁢          p                    -              (                  a          +                                    ∑                              i                =                1                            N                        ⁢                          xe2x80x83                        ⁢                                          b                i                            ⁢                              I                1                i                                              +                                    ∑                              i                =                1                            N                        ⁢                          xe2x80x83                        ⁢                                          c                i                            ⁢                              I                3                i                                                    )            
in which only one of the sums is used if only two images are used and correspondingly more sums are used if more images are used.